JP5473927B2 - Acetone production by fermentation from renewable raw materials via a novel metabolic pathway - Google Patents

Description

FIELD OF THE INVENTION The subject of the present invention is a novel biosynthetic method for the production of acetone that differs from the usual fermentation methods that separate the formation of ethanol and butanol, as well as the enzymes and nucleic acids used herein. .

Prior art ABE-methods in Clostridium The conventional ABE fermentation method, ie the production of acetone, butanol and ethanol by microorganisms has long been the second largest biotechnology process in the world following ethanol fermentation using yeast. Commercial ABE fermentation began in 1916 in the UK and in particular discovered the performance of Clostridium acetobutylicum producing acetone, butanol and ethanol as solvents by Heim Weitzmann. This process was used in the major worlds until the late 1950s, but was used in South Africa until 1981.

  Two main reasons stem from the fact that this process occurs as follows: on the one hand, chemical synthesis of acetone and butanol has always been promising, and on the other hand, the price of the substrate for fermentation is soaring. is there. In particular, the price of molasses has risen by using them as feed additives for cattle.

  Rising prices of petrochemical pre-products and new industrial possibilities in the field of microbial pathway engineering are new options for the development of high-performance strains and commercial fermentation processes that produce solvents such as acetone open.

  Conventional ABE-fermentation methods are based on the microorganisms Clostridium acetobutylicum and Clostridium beigerinkey. Both are gram positive and grow under severe anaerobic conditions. These microorganisms can be converted into mono-, di- and polysaccharides, with the main substrates used in fermentation being molasses and starch.

  The fermentation process with Clostridium acetobutylicum is divided into two phases. In the first phase, biomass formation involves the formation of acetate, butyrate and trace amounts of ethanol (the “acid production phase”). In the second phase, the so-called “solvent production phase”, acids are used to form the fermentation products acetone, butanol and ethanol (ABE). The products acetone, butanol and ethanol are formed in a ratio of about 3: 6: 1 in wild type Clostridium acetobutylicum. This product ratio varies significantly depending on the culture conditions selected (eg pH or nutrient supply) or the substrate used.

  The solvent-biosynthetic enzymes of acetone, butanol and ethanol are well washed and biochemically characterized (see FIG. 1; Duerre, P., and Bahl, H. 1996. Acetone / butanol / isopropanol). Microbial production, In: Biotechnology, Vol. 6, 2nd edition, M. Roeher, (ed.), VCH Verlaggesellschaft mbH, Weinheim, Germany. P. 229-268. Duerre, P. 1998. Acetone in Clostridium / New developments and new developments in butanol / isopropanol fermentation, Appl. Microbiol. Biotechnol. 49: 639-648). There is also a genomic sequence of Clostridium acetobutylicum (Noelling, J., Breton, G., Omelchenko, MV & 16 other authors (2001), comparative analysis with the genomic sequence of the solvent-producing bacterium Clostridium acetobutylicum, J. Bacteriol 183, 4823-4838).

  In recent years, a series of genetic tools have been developed that enable targeted pathway engineering. To date, there are three different replicons obtained stably (pIM13, pCBU2 and pAMβ1; Lee et al. (1992), vector creation, transformation and gene growth in Clostridium acetobutylicum ATCC 824, Ann. NY Acad Sci. 665: 39-51; Minton et al. (1993), Clostridium cloning vector [The clostridia and biotechnology (DR Woods); Hrsg, Butterworth-Heinemann. Stoneham, USA. 119-150, and erythromycin and thianpheny. Providing two antibiotic resistance markers for cole (Green and Bennett (1998), genetic manipulation of acid and solvent formation in Clostridium acetobutylicum ATCC 824, Biotechnol. Bioeng. 58: 215-21).

  In contrast, methods such as insertional inactivation or gene deletion are still not routinely feasible, and antisense-based inhibition of gene expression in this type of organism varies in efficiency, And never perfect (Desai and Papoutsakis (1999). Antisense RNA capture of metabolic engineering of Clostridium acetobutylicum, Appl. Environ. Microbiol. 65: 936-45; Tummala et al. (2003), Alcohol-producing Clostridium acetobutylicum Antisense RNA downregulation of coenzyme A transferase with alcohol-aldehyde dehydrogenase overexpression that preferentially induces fermentation, J. Bacteriol. 185: 3644-53). A series of publications describes metabolic techniques for both “solvent production” and “acid production” biosynthesis methods. Inactivation of the genes buk (butyrate kinase) or pta (phosphotrans-acetylase) results in a significant decrease in the concentration of butyrate or acetate (Green et al. (1996), genes in Clostridium acetobutylicum ATCC824 Gene amplification of acid formation pathway by inactivation, microbiology. 142: 2079-86 .; Harris et al. (2000), characterization of a recombinant strain of Clostridium acetobutylicum butyrate kinase inactivation mutant: Solvent production Novel phenomenological model and the need for butanol inhibition Biotechnol. Bioeng. 67: 1-11). Nevertheless, butyrate and acetate were formed from these mutants. This is because the enzymes acetate-kinase and phosphotransbutylylase are converted to the inactivated enzymes butyrate-kinase and phosphotransacetylase. Both strains form high concentrations of lactic acid that do not accumulate from wild-type Clostridium acetobutylicum as a reaction that, in some cases, accumulates pyruvate. Inactivation of the aldehyde / alcohol-dehydrogenase adE / aad results in a significant destruction of butanol formation and at the same time increases in the concentration of butyrate. When the adhE / aad gene is expressed on this mutant plasmid, the normal concentrations of butyrate and butanol in the wild type reappear. Interestingly, no formation of acetone could be detected in either AdhE / aad-mutant or strains with adhE / aad-complementarity. These phenomena are based on polar effects that act on two “downstream” sites of the adhE / aad localization gene. These two genes encode two subunits of coenzyme A-transferase that are heavy for the biosynthesis of acetone (Green, E. M, and Bennett, GN 1996. Aldehyde / alcohol dehydrogenase from Clostridium acetobutylicum ATCC 824. Gene inactivation, Appl. Biochem. Biotechnol. 57/58: 213-221). This is the first time that the formation of acetone and butyrate can be separated from each other by path engineering in Clostridium acetobutylicum. Further publications demonstrate these observations. This study examines microbial strains that have lost the ability to form acetone and butanol. This is because the 192-kb megaplasmid pSOL1 is missing. On this plasmid, most genes that form acetone and butanol are localized, and some are genes that synthesize ethanol (Cornillot, E., Nair, R., Papoutsakis, ET, and Soucaille, P 1997. The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 resides on a large plasmid whose loss leads to degenerierten of the strain. J. Bacteriol. 179: 5442-5447). This denatured strain formed only half as much ethanol as the wild type strain. These strains derived from Clostridium acetobutylicum ATCC 824 are referred to as M5 (resulting from chemical mutagenesis) and DG1 (obtained from wild-type multiple cultures). These are utilized as plasmid recipients, which include the protein coding sequences of coenzyme A-transferase subunits A and B (ctfA and ctfB), as well as the acetoacetate-decarboxylase (adc) gene, or butyraldehyde / butanol -Contributes to the gene for dehydrogenase (adhE / aad). The resulting strain produces only acetone or butanol (Mermelstein et al. (1993), metabolism of Clostridium acetobutylicum ATCC 824 to increase solvent production by enhancing acetone-forming enzyme activity using a synthetic operon. Engineering, Biotech. Bioeng. 42: 1053-1060 .; Nair, RV, and Papoutsakis, ET 1994. Expression of plasmid-encoded aad in Clostridium acetobutylicum M5 regenerating large amounts of butanol production, J. Bacteriol. 176: 5843 -5846; Cornillot, E., Nair, R., Papoutsakis, ET and Soucaille, P. 1997. A gene that forms butanol and acetone in Clostridium acetobutylicum ATCC 824, which is present in large plasmids and its deficiency results in strain degeneration J. Bacteriol. 179: 5442-5447).

  The titer measured for each solvent was essentially below the concentration of acetone and butanol in the wild type, where acetate and butyrate could accumulate to a total concentration of 240 mM. Ethanol formation could be produced again to the wild-type level with a plasmid that contributes to the adhE / aad gene.

  FIG. 2 describes a typical method of metabolism of acetone synthesis characterized by Clostridium. This method starts from acetyl-CoA and is the central metabolite formed in all microorganisms, regardless of which carbon source is metabolized or which metabolism is established. The required enzymes are: β-ketothiolase, two subunits of acetyl-CoA / butyryl-CoA-transferase, and acetoacetate-decarboxylase.

  Hetero-expression of enzymes from C. acetobutylicum in E. coli catalyzing the formation of acetone starting from acetyl-CoA has been shown to result in about 150 mM acetone formation in these organisms (acetoacetate-decarboxylase, Acetyl-CoA / butyryl-CoA-transferase and thiolase). However, a large amount of acetate (50 mM) is also incidentally produced as a drawback (Bermejo LL, NE Welker, ET Papoutsakis. 1998. Clostridium acetobutyricum in E. coli for acetone production and acetate detoxification ATCC 824 gene expression, Appl. Env. Microbiol. 64: 1079-1085). Here, another drawback is that acetone is produced only under aerobic conditions. This is because the redox equivalent generated when metabolizing glucose to acetyl-CoA cannot be oxidized again by E. coli under anaerobic conditions. In the method, CoA cleaved from acetoacetyl is transferred again to the receptor molecule by an enzyme that catalyzes the reaction.

Enzyme that separates acyl-CoA The enzyme capable of hydrolyzing acyl-CoA can be, for example, acyl-CoA-thioesterase or acyl-CoA-thiokinase. The main difference from acyl-CoA hydrolysis is that acyl-CoA-thioesterase or acyl-CoA-synthase / acetyl-CoA-thiokinase forms ATP or GTP in the case of kinases. This is because the hydrolysis of acetoacetyl-CoA has a higher ΔGo ′ value of −44 kJ compared to the hydrolysis of ATP (−31.8 kJ).

Acetoacetyl-CoA-hydrolase (EC 3.1.2.11)
This enzyme catalyzes the hydrolysis of acetoacetyl-CoA to acetoacetate and coenzyme A, but the CoA-molecules are not diverted simultaneously to acceptor molecules such as carboxylic acids.

Acyl-CoA-thioesterase (EC 3.1.2.18)
The protein YbgC from Haemophilus influenzae is an acyl-CoA-thioesterase (EC 3.1.2.18) specific for short-chain molecules, which accepts butyryl-CoA and β-hydroxybutyrate-CoA as substrates. These substrates have a Km value of 24 mM or 20 mM (Zhuang et al. (2002), YbgC protein encoded by the ybgC gene of the H. influenzae tol-pal gene cluster catalyzing the hydrolysis of acyl-coenzyme A thioesters. , FEBS Lett. 516: 161-3).

In B. subtilis, thioesterase II having the amino acid sequence SEQ ID NO: 1 and the corresponding cDNA- SEQ SEQ ID NO: 2 (TEII _srf) is being written, this peptide - non-ribosomal peptide to form a surfactin is an antibiotic Present in association with a synthase (Schwarzer D., HD Mootz, U. Linne, MA Marahiel. 2002. Denaturation of misprimed nonribosomal peptide synthase by type II thioesterase, PNAS 99: 14083-14088 acyl -CoA-synthetic enzyme / acyl-CoA-thiokinase (under AMP-formation; EC 6.2.1.2, under GDP formation; EC 6.2.1.10).

  The physical function of the two enzymes mentioned is in the acyl-CoA-synthesizing enzyme, but for example this enzyme catalyzes the reverse hydrolysis reaction while forming ATP, for example succinyl- in the tricarboxylic acid cycle. Something like CoA-thiokinase is known. To date, neither the above-mentioned enzyme classes capable of hydrolyzing these acetoacetyl-CoA while forming free coenzyme A with acetoacetate have been detected either in vitro or in vivo.

  Degradation of polyhydroxyalkanes by acyl-CoA-synthesizing enzymes under AMP-formation has been described for AcsA2 from Sinorhizobium meliloti (Aneja et al. (2002), in Shinorhizobium meriroti, L- Identification of an acetoacetyl coenzyme A synthase-dependent pathway utilizing (+)-3-hydroxybutyrate, J. Bacteriol. 184: 1571-7).

  A further purified enzyme from green bacterium could detect acyl-CoA-synthase / acyl-CoA-thiokinase specific for short-chain molecules (under AMP formation; EC 6.2.1.2). This enzyme accepts both butyryl-CoA and β-hydroxybutyryl-CoA and 2-ketobutyrate as substrates and has very low Km values of 10, 25 and 25 μM, respectively (Shimizu et al. (1981). Butyryl -CoA synthetase of Pseudomonas aeruginosa-purification and characterization. Biochem. Biophys. Res. Commun. 103: 1231-7).

  The object of the present invention was therefore to provide a new metabolic method simplified for the synthesis of acetone starting from acetyl-CoA.

DESCRIPTION OF THE INVENTION The invention described herein deals with improved acetone production by recombinant enzyme techniques: starting from acetyl-CoA, acetoacetate is produced via acetoacetyl-CoA, which continues to be It is converted into acetone and CO _2.

  The subject of the present invention is therefore a process for producing acetone as described in claim 1. In the method, the use of the enzyme according to claim 18 as well as the nucleic acid sequence according to claim 18 and the nucleic acid sequence according to claim 19 (the translation product of which is used to enable method step B of the method according to the invention). Use of the process of the invention).

  The method according to the invention has the advantage that in conventional systems acetoacetyl-CoA is converted to acetoacetate and the two subunit butyrate-acetoacetate-CoA-transferase is converted by one enzyme. Its activity can function as a monomer.

  Thereby, the process according to the invention has the further advantage that the yield of acetone in a given system is higher than the known butyrate-acetoacetate-CoA-transferase. Another advantage is that the acetate: acetone ratio during the production process is shifted in favor of acetone.

  A further advantage of the process according to the invention is that when using acetone synthesis separated from butanol and ethanol and thus microbial producers, they are not damaged by the concomitant alcoholic by-products. This results in a high product concentration in the medium, which in turn leads to an improved space time yield.

  Another advantage of producing acetone separately from butanol and ethanol is the most simplified fermentation protocol as well as easy isolation and processing of the product. This is because the alcoholic by-product does not have to be separated. This therefore requires less energy compared to product separation in conventional ABE-fermentation. Furthermore, the method according to the invention is particularly well characterized in that the method can be cultivated excellently both aerobically and anaerobically and has the most diverse possibilities for gene replication and therefore further improvements are known. It is advantageous to be able to carry out with the various microorganisms produced. Producing acetone efficiently from fermented raw materials by fermentation contributes to industrially environmentally friendly and resource-friendly production.

  The process for producing acetone according to the present invention and the use of said polypeptides and the nucleic acids for carrying out the process according to the present invention are exemplarily described hereinafter, but the present invention is not limited to this exemplary embodiment. In the event that a document is included within the scope of the description herein, the content shall be wholly belonging to the disclosure content of the present invention. A polypeptide means one having an arbitrary chain length. This is therefore interpreted for each protein, enzyme, in particular acetoacetate-CoA-hydrolase, acyl-CoA-thioesterase, acyl-CoA-synthase or acyl-CoA-thiokinase. “Spontaneous reaction” is interpreted as an ergonomic chemical reaction.

The present invention comprises the following method steps:
A: Acetyl-CoA is enzymatically reacted to acetoacetyl-CoA,
B: Acetoacetyl-CoA is enzymatically reacted to acetoacetate and CoA,
C: A method for producing acetone starting from acetyl-coenzyme A, comprising decarboxylating acetoacetate to acetone and CO ₂ , wherein said method does not divert coenzyme A to a receptor molecule in method step B It is characterized by that.

  In the method according to the invention, β-ketothiolase can be used in the enzymatic reaction in which acetyl-CoA is converted to acetoacetyl-CoA in method step A. Β-ketothiolase derived from Clostridium species, ie microorganisms from the genus Clostridium, in particular Clostridium acetobutylicum or Clostridium begerinki, are used. The enzymatic reaction in process step A has ketothiolase activity and is at least 90%, preferably at least 95%, more preferably up to 96, 97, 98, 99 or 100% with the amino acid sequence SEQ ID NO: 16. Advantageously catalyzed by polypeptides having amino acid sequences that are identical.

In the process according to the invention, a method step C, acetoacetyl -CoA it can be used acetoacetate decarboxylase reaction to acetone and CO _2. An acetoacetate decarboxylase derived from a Clostridium species, ie a microorganism from the genus Clostridium, in particular Clostridium acetobutylicum or Clostridium beigerinki is used. The enzymatic reaction in process step C has acetoacetate decarboxylase activity and is at least 90%, preferably at least 95%, more preferably 96, 97, 98, 99 or amino acid sequence SEQ ID NO: 18 or Advantageously, it is catalyzed by a polypeptide having an amino acid sequence that is up to 100% identical. Another embodiment of the process according to the invention is characterized in that the reaction in process step C takes place spontaneously.

  The method according to the invention for producing acetone is excellent in that coenzyme A is not diverted to the receptor molecule in process step B. This can be achieved by using an enzyme having acetoacetate-CoA-hydrolase activity. Surprisingly, similar enzymes whose activity has not been previously described, such as those selected from the group of acyl-CoA-thioesterase, acyl-CoA-synthase or acyl-CoA-thiokinase, The said subject can be solved similarly. Thus, an advantageous embodiment of the process according to the invention is that the enzymatic reaction in process step B is acetoacetate-CoA-hydrolase, acyl-CoA-thioesterase, acyl-CoA-synthase or acyl-CoA-thiokinase Catalyzed by

  In a preferred embodiment of the process according to the invention, the enzymatic reaction in process step B has acetoacetate-CoA-hydrolase activity and is at least 90%, preferably at least 95% with amino acid sequence SEQ ID NO: 1. Catalyzed by a polypeptide having an amino acid sequence that is up to%, more preferably up to 96, 97, 98, 99 or 100% identical. In a preferred embodiment of the process according to the invention, the enzymatic reaction in process step B has acetoacetate-CoA-hydrolase activity and is at least 90%, preferably at least 95% with amino acid sequence SEQ ID NO: 3. Catalyzed by a polypeptide having an amino acid sequence that is up to%, more preferably up to 96, 97, 98, 99 or 100% identical. In a particularly advantageous embodiment according to the invention, the enzymatic reaction in process step B has acetoacetate-CoA-hydrolase activity and is at least 90%, preferably at least 95% with amino acid sequence SEQ ID NO: 5. And more preferably catalyzed by a polypeptide having an amino acid sequence that is 96, 97, 98, 99 or 100% identical.

  Advantageously, the method is carried out with microorganisms. The microorganisms used in this method can themselves have the ability to synthesize acetone, or have the ability to form acetone by genetic pathway engineering. This is accomplished by catalyzing acetone synthesis starting from acetyl-CoA, independently of acetate, by increasing cell concentration or enzyme activity.

  Advantageously, the process according to the invention is carried out in a genetically altered microorganism. Such as, for example, Clostridium dimomonas sp., Escherichia sp., Salmonella sp., Rhodococcus sp. Derived from a genus selected from the genera Um, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Illustrative types of these genera are E. coli, Alkagenes eutrohus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas petitda, Enterococcus faecium, Enterococcus galinarium, Enterococcus faecalis, Bacillus subtilis Saccharomyces cerevisiae.

  In the method according to the present invention, Escherichia coli, Corynebacterium glutamicum, Clostridium species, Clostridium aceticum, Acetobacterium woody, Clostridium acetobutylicum, Clostridium begerinki, Yarrowia lipolytica, Saccharomyces species, Saccharomyces cerevisiae and Pitcha pastoris It is particularly advantageous to use a living body selected from “E. coli pUC19ayt” (see Examples 5 and 6).

  Recombinant microorganisms can be produced by recombinant gene methods known to those skilled in the art. In general, a vector having a heterologous gene can be infiltrated into a cell by an ordinary transformation method or transfection method. A suitable method can be found in Sambook et al. (Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory, 1989). The microorganism used can be a strain produced by mutagenesis or selection, by recombinant DNA methods, or by a combination of both methods. For mutagenesis, conventional in vivo-mutagenesis methods can be used with the use of mutagens, such as N-methyl-N′-nitro-N-nitrosoguanidine or UV light. it can. Furthermore, regarding in vitro mutagenesis, for example, treatment with hydroxylamine (Miller, JH: A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1992) or treatment with mutagenic oligonucleotides (TA Brown: Gentechnologie fuer Einsteiger, Spektrum Akademischer Verlag, Heidelberg, 1993) or polymerase chain reaction (PCR), eg Newton and Graham textbook (PCR, Spektrum Akademischer Verlag , Heidelberg, 1994).

  Further manuals for forming mutations are known from the prior art and known textbooks of gene and molecular biology, for example the textbook of Knipper ("Molekulare Genetik", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995 ), Winnacker ("Gene und Klone", VCH, publisher, Wienheim, Germany, 1990) or Hagemann ("Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986).

  When using the in vitro method, the genes described in the prior art are amplified using PCR starting from the whole isolated DNA of the wild type strain and optionally cloned in an appropriate plasmid vector. And subsequently this DNA is subject to mutagenesis. With regard to manuals for amplifying DNA sequences using polymerase chain reaction (PCR), those skilled in the art will find Gait textbooks: Oligonucleotide synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and Newton und Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994). Similarly, in-vitro- as described in a well-known textbook by Sambrook et al. (Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1989). Mutagenesis methods can also be used. A suitable method is, for example, “Quik Change Site-Directed Mutagenesis Kit” by Stratagene (La Jolla, USA) described by Papworth et al. (Strategies 9 (3), 3-4 (1996)). It is commercially available in the form of so-called “kits”.

  The subject of the invention is also a vector, in particular a plasmid, containing the polynucleotide used according to the invention and optionally replicated in bacteria. The subject of the present invention is also a recombinant microorganism transformed with the vector. In this case, the polynucleotide can be present under the action of one or more promoters. The subject of the present invention is also a recombinant microorganism transformed with said vector. In this context, the term “enhance” means an increase in the intracellular activity or concentration of one or more enzymes or proteins in the microorganism encoded by the corresponding DNA. This is because, for example, the copy number of one gene or multiple genes, one ORF or multiple PRFs is increased by at least one copy, and a strong promoter functionally binds to the gene or is highly active. This is done by using a single gene or allele or ORF encoding the corresponding enzyme or protein with or optionally a combination of these techniques. In E. coli, lac, tac and trp are typically called strong promoters. An open reading frame (ORF) is referred to as a protein or polypeptide that cannot incorporate any function according to the prior art, or a fragment of a nucleotide sequence that encodes or can encode a ribonucleic acid. After incorporating the function into a fragment of the nucleotide sequence, it is generally called a gene. An allele is generally taken to be an alternative form of a given gene. This form is characterized by differences within the nucleotide sequence.

  A gene product is generally a nucleotide sequence, ie a protein encoded by an ORF, gene or allele, or encoded ribonucleic acid. By enhancement, particularly overexpression techniques, the activity or concentration of the corresponding protein is generally relative to the wild-type protein or to the activity or concentration of the protein in the microorganism or parent strain that has not been recombined with the corresponding enzyme or protein. At least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to 1000% or 2000%. A non-recombinant microorganism or parent strain is interpreted as a microorganism that has been enhanced or over-expressed according to the invention. The gene or gene structure can be present in plasmids with various copy numbers, or can be integrated and amplified in the chromosome. Alternatively, overexpression of the gene can also be achieved by changing the medium composition and culture method.

  The subject of the present invention is a method for expressing a nucleic acid encoding a protein of an enzyme capable of carrying out process steps A to C as described above in a genetically modified microorganism.

The method according to the invention comprises at least one nucleic acid a in which the microorganism encodes a protein of at least one polypeptide.
(Amino acid sequence having ketothiolase activity and having the amino acid sequence SEQ ID NO: 16 at least 90%, preferably at least 95%, preferably at least 95%, 97%, 98%, 99% or 100% identical. Have)
A nucleic acid b encoding a protein of at least one polypeptide
(Acetoacetate-CoA-hydrolase, acyl-CoA-thioesterase, acyl-CoA-synthase or acyl-CoA-thiokinase, or has acetoacetate-CoA-hydrolase activity and amino acid sequence Having an amino acid sequence identical to SEQ ID NO: 1 up to at least 90%, preferably at least 95%, preferably up to 96%, 97%, 98%, 99% or 100%, or acetoacetate-CoA-hydrate An amino acid sequence having a degrading enzyme activity and having the amino acid sequence SEQ ID NO: 3 up to at least 90%, preferably at least 95%, preferably at least 96%, 97%, 98%, 99% or 100% identical Or having acetoacetate-CoA-hydrolase activity and amino acid sequence SEQ ID NO: 5 and at least 90%, preferably less Least 95%, preferably 96%, the 97%, 98%, 99% or having an amino acid sequence identical up to 100%) and nucleic acid encoding a protein of at least one polypeptide c
(Has acetoacetate decarboxylase activity and is at least 90%, preferably at least 95%, preferably 96%, 97%, 98%, 99% or 100% identical to amino acid sequence SEQ ID NO: 18 (Has an amino acid sequence)
Wherein the nucleic acids a, b and c are used to ensure the expression of the polypeptide in the microorganism.

In this regard, the following:
aa DNA sequence described in sequence 17 or its complementary strand,
bb a DNA sequence that hybridizes under stringent conditions to the DNA sequence described in aa;
It is advantageous to use a nucleic acid a selected from a DNA sequence that hybridizes to the DNA sequence defined by aa and bb without the degeneracy of the cc gene code.

As nucleic acid c, the following:
dd DNA sequence described in sequence 19 or a complementary strand thereof,
ee DNA sequence that hybridizes to the DNA sequence described in dd under stringent conditions;
It is advantageous to use a DNA sequence that hybridizes to the DNA sequence described in dd and ee without the degeneracy of the genetic code.

As nucleic acid b, the following:
gg DNA sequence described in sequence 2 or its complementary strand,
hh a DNA sequence that hybridizes under stringent conditions to the DNA sequence described in gg;
ii It is advantageous to use those selected from DNA sequences that hybridize to the DNA sequences described in gg and hh without the degeneracy of the genetic code, or more preferably:
jj DNA sequence described in sequence 4 or its complementary strand,
a DNA sequence that hybridizes to the DNA sequence described in jj under kk stringent conditions;
It is advantageous to use those selected from DNA sequences that hybridize to the DNA sequences described in jj and kk, without the degeneracy of the ll gene code, or particularly preferably:
mm DNA sequence described in sequence 6 or its complementary strand,
nn DNA sequence that hybridizes to the DNA sequence described in mm under stringent conditions;
It is advantageous to use those selected from DNA sequences that hybridize to the DNA sequences described in mm and nn without the degeneracy of the oo gene code.

  In a preferred embodiment of the method according to the invention, the nucleic acids a, b and c are on a single nucleotide chain allowing the expression of all three gene products.

Nucleic acids a, b and c are on one nucleotide chain and this is:
A plasmid pSKatt having the nucleic acid sequence according to SEQ ID NO: 7,
A plasmid pKSatt having the nucleic acid sequence according to SEQ ID NO: 8,
Plasmid pUC19att having the nucleic acid sequence according to SEQ ID NO: 9,
Plasmid pUC18att having the nucleic acid sequence according to SEQ ID NO: 10,
Plasmid pUC19ayt having the nucleic acid sequence according to SEQ ID NO: 11
It is particularly advantageous to select from the group having

  Accordingly, the subject of the present invention is the plasmid pSKatt having the nucleic acid sequence according to SEQ ID NO: 7, the plasmid pKSatt having the nucleic acid sequence according to SEQ ID NO: 8, the plasmid pUC19att having the nucleic acid sequence according to SEQ ID NO: 9, and the plasmid pUC18att having the nucleic acid sequence according to SEQ ID NO: 10. And a nucleic acid having plasmid pUC19ayt having the nucleic acid sequence according to SEQ ID NO: 11.

  Another subject of the invention is the use of acyl-CoA-thioesterase, acyl-CoA-synthesizing enzyme or acyl-CoA-thiokinase, which enzymatically reacts acetoacetyl-CoA with acetoacetate and coenzyme A. Because of the high substrate specificity of most enzymes, it was unexpected and unpredictable for those skilled in the art that these classes of enzymes would accept acetoacetate-CoA as a substrate.

It is advantageous to use a polypeptide having an acyl-CoA-thioesterase activity, an acyl-CoA-synthesizing activity, or an acyl-CoA-thiokinase activity in which acetoacetyl-CoA is enzymatically reacted with acetoacetate and coenzyme A. The polypeptide is acetoacetate-CoA-hydrolase, acyl-CoA-thioesterase, acyl-CoA-synthase or acyl-CoA-thiokinase, or up to at least 90% with amino acid sequence SEQ ID NO: 1. Preferably having at least 95%, more preferably having an amino acid sequence identical to 96%, 97%, 98%, 99% or 100%, or at least 90%, preferably with amino acid sequence SEQ ID NO 3 Up to at least 95%, more preferably up to 96%, 97%, 98%, 99% or 100% An amino acid sequence having an amino acid sequence or being identical to amino acid sequence SEQ ID NO: 5 by at least 90%, preferably by at least 95%, more preferably by 96%, 97%, 98%, 99% or 100% Have

  The subject of the present invention is therefore also the use of at least one of the nucleic acids b selected from the group gg to oo for utilizing the gene product for the reaction of acetoacetyl-CoA to acetoacetate and CoA.

  In the following examples, the present invention will be described by way of example, and the scope of the present invention is derived from the entire description and claims, but is not limited to the embodiments given in the examples.

FIG. 1 is a diagram representing the biosynthesis of acetone, butanol and ethanol. FIG. 2 is a diagram showing the biosynthesis of acetone. Figure 3 is a diagram showing the expression of TEII _srf the inside E.Coli M15. Figure 4 is a diagram showing the purification of TEII _srf from E.Coli. 5, with respect to the substrate acetyl-CoA and acetoacetyl-CoA, line Weaver, Burg calculates a Km value of TEII _srf - a diagram representing the FIG. FIG. 6 is a diagram showing the expression of AACS (DE3) in E. coli BL21. FIG. 7 shows the purification of AACS from E. coli. FIG. 8 shows the expression and purification of YbgC. Figure 9 is a diagram representing genes in plasmid pSK adcs, a picture of successive cloning TEII _srf and thlA. FIG. 10 shows the creation of the pUC19-structure. FIG. 11 is a diagram showing acetone formation and acetate formation in various expression vectors in E. Coli HB101 (5 ml medium). FIG. 12 is a diagram showing acetone formation and acetate formation in 100 ml medium of E. Coli HB101 containing pUC19ayt (YbgC) and pUC19act (control CtfA / B).

Example Example 1: Bacillus subtilis-derived thioesterase II (TEII _srf)
This enzyme was mixed with non-ribosomal peptide-synthetic enzyme in Bacillus subtilis ATCC 21332 to form surfactin (peptide-antibiotic). Schwarzer, etc., cloned genes corresponding in the plasmid pQE60 which mediates fusion between the target protein with a His-Tag at the C-terminus and (pTEII _srf), and it was possible to purify this protein (28 kDa) ( Schwarzer D., HD Mootz, U. Linne, MA Marahiel. 2002, regeneration of misprimed non-ribosomal peptide synthase by type II thioesterase, PNAS 99: 14083-14088). In subsequent tests, the protein was hydrolyzed with acetyl-CoA and propionyl-CoA as substrates.

. Plasmid PTEII _srf having SEQ ID NO: 13, Schwarzer D, Mootz HD, Linne U, playback Marahiel MA (non-ribosomal peptide synthetase which is mispriming by type II thioesterase, Proc Natl Acad Sci USA 2002 Oct 29; 99 (22): 14083-8).

Protein expression in E. Coli M15 in LB _AMP -medium was performed at 30 ° C. and 150 rpm and induced with 1 μM IPTG at an optical density of 0.6-0.8 (OD _{600 nm} ). After an additional 2 hours, the medium was collected. The probe obtained during growth confirmed successful protein expression. FIG. 3 shows a 12% SDS-polyacrylamide gel loaded with 15 μl of crude extract per lane after Gramercy staining; 1: before induction, 2: after 1 hour of induction, 3 : 2 hours after induction.

The predictive activity measurement measured with the free SH-group by the DTNB-assay [(5,5′-dithiobis (2-nitrobenzoic acid)] required the purification of the protein, which was FPLC-supported TEII. _This was done by increasing the imidazole gradient by metal chelate affinity chromatography on Ni ²⁺ -NTA-agarose with the fused His-Tag of srf.

For this purpose, cells were first obtained (5000 × g, 15 minutes, 4 ° C.), and an undried weight of 3 ml / g was suspended in a cytolysis buffer (50 mM HEPES, 300 mM NaCl, pH 7.8) And optionally stored at -20 ° C. Cells were lysed by sonication (Sonicator Bandelin Sonopuls, Bandelin Berlin) and a crude extract was obtained by centrifugation (30000 × g, 30 min, 4 ° C.). Purification was performed on an FPLC-apparatus (Pharmacia Biotech GmbH). 3.5 ml crude in a correspondingly prepared and equilibrated (50 mM HEPES, 300 mM NaCl, 30 mM imidazole, pH 7) Ni ²⁺ -NTA-agarose column (Qiagen Superflow, column bed volume 5 ml) according to the manufacturer's instructions. The extract was loaded. Subsequently, the column was washed with 50 ml of equilibration buffer. Elution was performed with a linear imidazole-gradient exceeding 50 ml (30-300 mM imidazole, 300 mM NaCl, pH 7 in 50 mM HEPES).

In the upper range of FIG. 4, the FPLC-elution profile in Ni ²⁺ -NTA-agarose (Qiagen Superflow, column bed volume 5 ml Bettvolume 5 ml) is shown. Here, the program is as follows: wash 0-50 ml, elution 30-300 mM imidazole 50-100 ml (linear gradient) and fraction size every 1 ml; lower range is 12% SDS-PAGE- stained with Coomassie Gel is depicted. Here, 23 to 31 elution fractions and 1 μg of protein per lane are plotted.

  The obtained fractions were collected and used for activity measurement. Acetoacetyl-CoA was used as a substrate and acetyl-CoA was used for comparison. In the DTNB-assay, a sulfhydryl group at the terminal of CoA released to acetoacetate or acetate by an enzymatic reaction was reacted with DTNB to give a colored product. This could be determined by photometric measurement at 412 nm.

The following Km-values were determined: 2 · 10 ⁻⁴ mol · 1 ⁻¹ (0.2 mM) for acetyl-CoA and 7.7 · 10 ⁻⁴ mol · 1 ⁻¹ (0.77 mM) for acetoacetyl-CoA. . Therefore, TEII _srf had high substrate affinity for acetyl-CoA. 5 shows, with respect acetyl -CoA and acetoacetyl -CoA a substrate, the line Weaver, Burg calculates a Km value of TEII _srf - Figure is shown.

Example 2: Acetacetyl-CoA-synthesizing enzyme (AACS) from Sinorhizobium meliloti
The acetoacetyl-CoA-synthesizing enzyme (AACS) having the amino acid sequence SEQ ID NO: 3 and the corresponding cDNA-sequence SEQ ID NO: 4 derived from Sinorhizobium meriroti catalyze the reaction of acetoacetate and coenzyme A while consuming ATP. Acetoacetyl-CoA and AMP. However, the reverse reaction was of interest within the scope of the present invention.

The corresponding gene acsA2 was cloned in the expression vector pET30Xa / LIC as described in Aneja, PR Dziak, GQ Cai, TC Charless, 2002. The identification of an acetoacetyl coenzyme A synthase-dependent pathway utilizing L-(+)-3-hydroxybutyrate within the Sinorhizobium meriroti is disclosed in J. Bacteriol. 184: 1571-1577. The plasmid “pRD112” thus obtained mediates the fusion of the N-terminus of the protein of interest with the His-Tag, which further allows purification by affinity chromatography on Ni ²⁺ -NTA-agarose.

E. Coli BL21 (DE3) —Expression of the protein in the cells was performed at 37 ° C. and 180 Upm. The medium was induced with 1 mM IPTG at an optical density of 0.5-0.6 (OD _{600 nm} ) and subsequently incubated for 3 hours. Probes obtained during growth were detected from successful expression of the protein (approximately 72 kDa).

  FIG. 6 describes a 7.5% strength SDS-PAGE-gel after Coomassie staining; 15 μg of crude extract per lane (fast cell lysis) was plotted. 1: Before induction, 2: 1 hour after induction, 3: 2 hours after induction.

Purification following the ones TEII _srf, was performed by a metal chelate affinity chromatography and 20~250mM Imidazole gradient in Ni ²⁺ -NTA-agarose (Figure 7A). In this case, two peaks (1 and 2) appeared and the protein content of those fractions was examined. Subsequent analysis is shown in a 7.5% concentration SDS-PA-gel. Especially in the first peak, the appearance of a further band was at about 55 kDa. Since these bands were significantly weaker or did not occur at all in the second peak, the corresponding fractions of these peaks were combined and used for activity measurements. Again acetoacetyl-CoA was used as substrate and acetyl-CoA was used in the DTNB-assay for comparison. As for the measurement of Km-value, a value of 7 · 10 ⁻⁴ mol · 1 ⁻¹ (0.7 mM) was obtained with acetyl-CoA as a substrate. On the other hand, the value measured with acetoacetyl-CoA was insufficient for the measurement of Km-value. However, since the specific activity of the enzyme and acetoacetyl-CoA was only 0.0038 μmol · min ⁻¹ · mg ⁻¹ , AAC derived from Sinorhizobium meriroti was used for cloning combining the corresponding gene and the clostridial gene. Except for further use. FIG. 7 shows the purification of AACS from E. coli, the upper range showing the FPLC-elution profile in Ni ²⁺ -NTA-agarose (Qiagen Superflow, column bed volume 5 ml). The program is as follows: 0-100 ml washed, 100-150 ml eluted with 20-250 mM imidazole (linear gradient) and fraction size per ml; lower range is after silver staining 7.5% SDS-PAGE-Gel is shown. Here, the selected peak 1 and peak 2 elution fractions are 1 μg protein per lane. RE: Crude extract, DL: Pass.

Example 3: Acyl-CoA-thioesterase YbgC from Haemophilus influenzae
The YbgC protein from Haemophilus influenzae having the amino acid sequence SEQ ID NO: 5 and the corresponding cDNA-sequence SEQ ID NO: 6 was similar to the corresponding protein from E. Coli. This belongs to the so-called Tol-Pal-system as a cytoplasmic protein. This is widely propagated within Gram-negative bacteria, is important for maintaining cell wall integrity, and in some cases has a function in transporting substances through the surrounding cytoplasm. On the other hand, the possible relationship between the function of YbgC in H. influenzae and the function of the Tol-Pal-system has not been published so far. However, a publication by Zhuang et al. (2002) describes the testing of the catalytic function of this protein and the analysis of thioesterase activity. In this case, hydrolysis of single-chain aliphatic acyl-CoA-esters such as propionyl-CoA and butyryl-CoA (Km value 11-24 mM) with YbgC has been shown (Zhuang Z., F. Song, BM Martin, D. Dunaway-Mariano. 2002. YbgC protein encoded by the ybgC gene of the H. influenzae tol-pol-gene cluster catalyzes the hydrolysis of acyl-coenzyme A thioester, FEBS Lett. 516: 161- 163). In order to detect in-vitro activity of acetyl-CoA and acetoacetyl-CoA within the scope of this purpose, purification of a new protein is a prerequisite. Starting from genomic DNA, the gene was cloned in another expression system, namely the NEB (Frankfurt An Main) IMPACT ^™ (Intein Mediated Purification with an Affinity Chitin-binding-Tag) system. The advantage lies in that the method allows simple purification of the recombinant protein in its original form, ie without a tag.

  Cloning of ybgC in the vector pTYB1 (NEB Frankfurt an Main) allows expression and subsequent purification of the acyl-CoA-thioesterase YbgC from E. coli.

The gene ybgc was amplified by genomic DNA PCR using primers ybgcTYB1Ndefw (ATA TAC ATA TGT TGG ATA ATG GCT TTT C) and ybgcTYB1Xhorev (TCC GAA CTC GAG TTT TAA GTG ATG). In this case, the primer mediates the incorporation of an NdeI-cleavage site at the 5′-end of the amplified fragment or an XhoI-cleavage site at the 3′-end. Taq-master mix (Qiagen, Hilden) was used under the following conditions according to the manufacturer's instructions:

A fragment of the expected size of about 430 Bp was obtained. This was first subcloned in the pJET-vector (Fermentas, St. Leon-Rot) according to the manufacturer's instructions (pJet_ybgcTYB, 410Bp). The ybgc-fragment was subsequently excised from this plasmid with restriction endonucleases NdeI and XhoI and gel eluted (obtained according to the gel extraction kit, peqlab Biotechnologye GmbH, manufacturer's instructions). The vector pTYB1 (7477Bp) was similarly cut with NdeI and XhoI (7439Bp) and subsequently cut using the Rapid Ligation Kit (Fermentas GmbH, St. Leon-Rot) according to the manufacturer's instructions and gel eluted Ligated with the ybgc fragment. For every 10 μl of ligation reaction, 100 μl of CaCl ₂ -competent E. ColiXL1-B-cells were used for transformation. The resulting clone was subsequently grown in LB-ampicillin-liquid medium and plasmid-DNA was isolated. Isolated plasmids were checked by restriction analysis (NdeI / XhoI) and sequenced (Agowa GmbH, Berlin). The resulting plasmid was marked with pTYBybgc (SEQ ID NO: 15) and used in E. Coli BL21 DE3 for further transformation.

Expression is performed in E. Coli BL21 (DE3) in LB _AMP -medium with 0.5 mM IPTG at 37 ° C. until induced at an OD _{600 nm} of about 0.5, after which the medium is incubated at 20 ° C. and 150 rpm. Further incubation for 6 hours. The fusion protein (about 70 kDa) examined for heterogeneity bound to chicken. Addition of DTT induced a cleaved portion of the target protein (about 15 kDa) that limited the change in morphology and could subsequently be eluted. FIG. 8 shows an SDS-PAGE-gel, and the upper range shows SDS-PAGE obtained by analyzing the expression of YbgC. This is a gradient-SDS-PA-gel with 10-20% concentration after Coomassie staining. This was loaded with 15 μl of crude extract (fast cell lysis) per lane. 1: Before induction, 2, 3: 6 hours after induction. The lower part describes the process of purification of YbgC on a chitin column in a 10-20% gradient-SDS-PA-gel after silver staining. This was loaded with 2 μl protein (fast lysis) per lane. -RE: crude extract, DL: passage, W: wash fraction, elution: protein-containing elution fraction (fraction size: every 1 ml).

In the elution fraction, in addition to the desired protein of 15 kDa, a fusion protein band and a split intein domain were shown, which were used for in vitro activity measurement in the DTNB-assay. The decision to calculate the Km-value was uncertain (data not shown), but with respect to acetyl-CoA as the substrate, the Km value of 5.3 · 10 ⁻⁴ mol·l ⁻¹ (0.53 mM), acetoacetyl- For CoA, a Km value of 1.4 · 10 ⁻⁴ mol·l ⁻¹ (0.14 mM) was calculated. Moreover, a direct comparison was able to determine the enzyme's high substrate affinity for acetoacetyl-CoA. This result indicates that YbgC can be added to the cloning for further testing.

Example 4: Cloning strategy to construct an expression vector
In order to produce acetone in E. coli, the gene thlA from C. acetobutylicum (the amino acid sequence of SEQ ID NO: 16 and the corresponding cDNA-gene product having the sequence SEQ ID NO: 17, thiolase A) and adc (of SEQ ID NO: 18 A suitable acyl-CoA-thioesterase or acyl-CoA-synthetase / acyl-CoA-thiokinase gene clone together with the amino acid sequence and the corresponding cDNA-sequence gene product having the sequence number 19 (acetoacetate-decarboxylase) Made. For this purpose, plasmids pSK and pKS were first selected. This is a cloning vector that is substantially different from the direction of the multiple cloning site (MCS). Each MCS is within the existing lacZ'-gene sequence, which encodes the N-terminal fragment of β-galactosidase and allows blue-white-screening of recombinant plasmids. For testing within the scope of the project, it is important to express the cloned gene under the control of an inducible lac-promoter. For this purpose, a vector psK was prepared. Furthermore, mutants are produced by insertion into plasmid pKS. The gene should then be tested under the control of a constructive thiolase-promoter derived from C. acetobutylicum.

  Cloning of each gene in the vector is performed sequentially. For this purpose, oligonucleotides for gene amplification were first designed with the corresponding cleavage sites inserted and the polymerase chain reaction was carried out. All fragments were amplified and cloned into the vector as described above. The strategy for pSK and the restriction cleavage sites used are shown schematically in FIG. Similarly, this was performed using plasmid pKS.

The cloning process is disclosed in detail below:
Cloning of genes adc, teII, thIA in plasmid pKS:
Gene adcs (acetoacetate from Clostridium acetobutylicum - decarboxylase), (thioesterase II from Bacillus subtilis) TEII _srf and
The purpose was to clone thIA (thiolase from C. acetobutylicum) together with one vector and subsequently express it in E. coli.

The acetoacetate-decarboxylase gene adc was amplified by PCR of the plasmid pDrive_adc (SEQ ID NO: 20) using primers AdcXhofwneu (CAT GCT CGA GAC GCG TTA CGT ATC) and AdcAparevneu (GAT GGG GCC CTG AAT TCT ATT ACT TAA G). It was. In this case, the primer mediated insertion of an XhoI-cleavage site at the 5′-end of the amplified fragment or an ApaI-cleavage site at the 3′-end. The polymerase GoTaq (Promega GmbH, Mannheim) was used under the following conditions while adding 4 mM MgCl ₂ according to the manufacturer's instructions:

A fragment of the expected size of about 800 Bp was obtained. This is gel eluted (gel extraction kit; peqlab Biotechnologie GmbH, obtained according to manufacturer's instructions) and subsequently cleaved with restriction endonucleases XhoI and ApaI (788Bp) as in the case of plasmid pKS and the vector added (Antarctic phosphatase, New England Biolabas GmbH, Frankfurt am Main, according to the manufacturer's instructions). Subsequently, ligation of the vectors and fragments thus treated was performed using T4-ligase (New England Biolabas GmbH, Frankfurt am Main) according to the manufacturer's instructions. For every 10 μl of ligation reaction, 100 μl of CaCl ₂ -competent E. ColiXL1-B-cells were used for transformation. For each reaction, 100 μl CaCl ₂ -competent E. ColiXL1-B-cells are transferred to a pre-chilled 1.5 ml reaction vessel, and 10 μl ligation reaction is added to this and mixed carefully and the reaction The object was incubated on ice for 30 minutes. Subsequently, heat shock is applied at 42 ° C. for 90 seconds, and the cells are then placed on ice for 2 minutes, to which 0.5 ml of pre-warmed LB-medium is added, and the reaction is shaken at 37 ° C. for 60 minutes. Incubated. 100-300 μl of each reaction was plated on LB-ampicillin medium and incubated overnight at 37 ° C. The resulting clones were subsequently grown in LB-ampicillin liquid medium and plasmid DNA was isolated according to the following protocol: For rapid isolation of plasmid-DNA, "Qiagen Mini-Kit" (Qiagen , Hilden) was used following Birnboim and Doly (Birnboim, HC, J. Doly. 1979. A rapid alkaline extraction process for screening recombinant plasmid DNA. Nucleic Acids Res. 7: 1513-1523). Isolated plasmids were examined by restriction analysis (ApaI / XhoI) and subsequently positive clones were sequenced (Agowa GmbH, Berlin). This plasmid was marked with pKSadc and used in subsequent cloning steps.

Thioesterase II gene thII _srf, was amplified by PCR of the plasmid PTEII _srf using primers ThIISalfw (CTT TTG TCG ACG GAT AAC AAT TTC ACA CAG A) and ThIIXhorev (CTA TCA ACT CGA GTC CAA GCT CAG CTA ATT AA). In this case, the primer mediates the incorporation of a SalI-cleavage site at the 5′-end of the amplified fragment or an XhoI-cleavage site at the 3′-end. Synergy-polymerase (GeneCraft GmbH., Ludinghausen) was used according to the manufacturer's instructions under the following conditions:

A fragment of the expected size of about 850 Bp was obtained. Gel elution (gel extraction kit; peqlab Biotechnologie GmbH, obtained according to manufacturer's instructions) and subsequent digestion with restriction endonucleases SalI and XhoI (831Bp) as in the case of plasmid pKSadc and additional vector (Shrimp alkaline phosphatase SAP, Fermentas GmbH, St. Leon-Rot, according to the manufacturer's instructions). Subsequently, ligation of the vectors and fragments thus treated was performed using a rapid ligation kit according to the manufacturer's instructions (Fermentas GmbH, St. Leon-Rot). For every 10 μl of ligation reaction, 100 μl of CaCl ₂ -competent E. ColiXL1-B-cells were used for transformation. 100-300 μl from each reaction was plated on LB-ampicillin-medium and incubated overnight at 37 ° C. The resulting clone was subsequently grown in LB-ampicillin liquid medium and plasmid DNA was isolated. Isolated plasmids were examined by restriction analysis (XhoI / SalI) and subsequently positive clones were sequenced (Agowa GmbH, Berlin). This plasmid was marked with pKSadcte and used in subsequent cloning steps.

  The thioesterase gene teIA was amplified including the thiolase-promoter by PCR of genomic DNA from C. acetobutylicum.

Isolation of chromosomal DNA from C. acetobutylicum:
Isolation of chromosomal DNA from C. acetobutylicum was in principle performed by Bertram (Bertram, J. 1989, Development of Clostridium acetobutylicum DNA transfer and transposon mutagenesis system, dissertation, University of Gottingen) .

For this, cells were grown in 2 × YTG-anaerobic medium and harvested at an OD ₆₀₀ of about 1.2. The cells were pelleted by centrifugation (5 minutes, 5000 × g, 4 ° C.). The cell pellet was washed 3 times with 10 ml of 1 × TAE buffer (supplemented with 10% [w / v] saccharose) and then stored at −20 ° C. DNA was obtained from 90 ml of the cell suspension thus treated as follows:
1. 1. Suspend the pellet in 3.8 ml of 1xTAE containing sucrose. 2. Add 1 ml of lysozyme-RNase solution. Incubation: 30 minutes, 37 ° C
4. Add 500 μl of 0.5 M EDTA (pH 8.0), 40 μl Tris-HCl (pH 8.0), 30 μl SDS (10% [w / v])
5. Incubation: 10 minutes, 37 ° C
6). 6. Add 200 μl of proteolytic enzyme K solution Incubation: 2 hours at 37 ° C
8. Add 1.4 ml of 8.5M sodium perchlorate. Add 7.3 ml of chloroform-isoamyl alcohol (24: 1 [v / v]) to ice. Centrifugation: 10 minutes, 5000 × g, 4 ° C.
11. Remove upper aqueous phase 12. 13. Second repeat of steps 9-11 13. Precipitation of DNA from aqueous phase by addition of 1 volume isopropanol Incubation: 5 minutes, room temperature 15. Centrifugation: 20 minutes, 16000 × g, 4 ° C.
16. 17. Dry pellet at room temperature for up to 2 hours Suspend pellet in 2 ml TE-buffer 18. 20. Add 200 μl of proteolytic enzyme K-solution Incubation: overnight at 37 ° C. 20. Increase volume to 4 ml with A. dest. Add 600 μl of 21.3 M sodium acetate (pH 5.2)
22. Extraction with chloroform-isoamyl alcohol three times (see steps 9 to 11)
DNA precipitation by addition of 23.1 volumes of isopropanol 24. Incubation: 5 minutes, room temperature 25. Centrifugation: 20 minutes, 16000 × g, 4 ° C.
26. Wash pellet twice with 96% (v / v) ethanol (pure ice cold)
27. Dry pellets at room temperature, max 2 hours 28. Suspend pellet in 200 μl TE.

Primers used in PCR, PthlAXmafw (CAT GAT TTC CCG GGG GTT AGC ATA TG) and PthlASalrev (CAG AGT TAT TTT TAA GTC GAC TTT CTA GCA C), are at the 5′-end of the fragment amplified from the XmaI-cleavage site, or Mediates the incorporation of a SakI-cleavage site at the 3′-end. Taq-master mix (Qiagen, Hilden) was used under the following conditions according to the manufacturer's instructions:

A fragment of the expected size of about 1400 Bp was obtained. This was first subcloned into pJET-vector (Fermentas, St. Leon-Rot) according to the manufacturer's instructions (pJet_PthlA). Subsequently, the thlA-fragment was cut from this plasmid again with restriction endonucleases XhoI and Cfr9I (1397Bp) and gel eluted (obtained according to the manufacturer's instructions, gel extraction kit, peqlab Biotechnologye GmbH). Plasmid pKSadcte was similarly treated with XhoI and Cfr9I and additionally dephosphorylated (Shrimp alkaline phosphatase SAP, Fermentas GmbH, St. Leon-Rot, according to manufacturer's instructions). This was followed by ligation of the treated vectors and fragments according to the manufacturer's instructions using a rapid ligation kit (Fermentas GmbH, St. Leon-Rot). For every 10 μl of ligation reaction, 100 μl of CaCl ₂ -competent E. ColiXL1-B-cells were used for transformation. Subsequently, the resulting clones were grown in LB-ampicillin-liquid medium and plasmid-DNA was isolated. Isolated plasmids were examined by restriction analysis (SalI / Cfrl9I) and positive clones were subsequently sequenced (Agowa GmbH, Berlin).

  The plasmid having SEQ ID NO: 8 was marked with pKSatt and used in various E. coli strains for further transformation, which was subsequently examined for the formation of acetone.

The plasmid pSK in a gene adc, teII _srf, cloning of thlA:
In order to clone the three genes in the vector pSK, initially already adcs-TEII _srf present in pKS - by restriction fragment, cut with ApaI and SalI (1625Bp), and gel eluted (Gel Extraction Kit; Peqlab Biotechnologie GmbH, obtained according to manufacturer's instructions), and ligated in similarly cut and dephosphorylated pSK-vector (SAP, Fermentas GmbH, St. Leon-Rot, according to manufacturer's instructions). For this, a quick ligation kit (New England Biolabs GmbH, Frankfurt am Main) was used according to the manufacturer's instructions. Subsequently, in order to examine the resulting clones, they were grown in LB-ampicillin-liquid medium and plasmid-DNA was isolated. The isolated plasmid was examined using restriction analysis (ApaI / SalI). The plasmid thus obtained was marked with pSKadcte and further used in the following cloning step.

The thiolase gene thlA was amplified by PCR of the plasmid pDrive_thl (SEQ ID NO: 21) using primers ThlAXmafw (GTC GAC CCG GGT CAA AAT TTA GGA G) and ThlASalrev (GCT TGT CGA ATT CAG ATC AGA G). In this case, the primer mediates the incorporation of an XmaI-cleavage site at the 5′-end of the amplified fragment or a SalI-cleavage site at the 3′-end. Taq-master mix (Qiagen, Hilden) was used under the following conditions according to the manufacturer's instructions:

A fragment of the expected size of about 1250 Bp was obtained. This was first subcloned into pJET-vector (Fermentas, St. Leon-Rot) according to the manufacturer's instructions (pJet_thlA). Subsequently, the thlA-fragment was cut from this plasmid again with restriction endonucleases XhoI and Cfr9I (1243Bp) and gel eluted (obtained according to the manufacturer's instructions, gel extraction kit, peqlab Biotechnologie GmbH). Plasmid pSKadcte was similarly marked with XhoI and Cfr9I and additionally dephosphorylated (Shrimp alkaline phosphatase SAP, Fermentas GmbH, St. Leon-Rot, according to manufacturer's instructions). Subsequently, the ligation of the vectors and fragments thus treated was carried out using a quick ligation kit (New England Biolabs GmbH, Frankfurt am Main) according to the manufacturer's instructions. For every 10 μl of ligation reaction, 100 μl of CaCl ₂ -competent E. ColiXL1-B-cells were used for transformation. The resulting clone was subsequently grown in LB-ampicillin-liquid medium and the plasmid-DNA was isolated. Isolated plasmids were examined by restriction analysis (SalI / Cfrl9I) and subsequently positive clones were sequenced (Agowa GmbH, Berlin). The plasmid with SEQ ID NO: 7 was marked with pSKatt and used in various E. coli strains for further transformation.

Based on the pUC18-construct (pUCadc_ctfA / B_thlA; see FIG. 10) having the SEQ ID NO: 14 in which the clostridial genes adc, ctfA / B and thlA have already been cloned, are present, while additional mutants in the vector pUC19 The gene cassette was expressed under the control of the lac promoter. Moreover, continued could be cut to specific genes ctfA / B, insert a gene TEII _srf or ybgC after inserting the appropriate cleavage site in fragment (Figure 9). In addition, in the pUC18-construct, the original thlA-fragment was removed and replaced with a thlA-fragment containing the "thl-promoter sequence" upstream.

FIG. 10 illustrates the generation of the pUC19-construct; the upper diagram schematically illustrates the method for cloning the gene cassette adc / ctfAB / thlA in the plasmid pUC19, the lower one is the plasmid pUC19act. starting from one in which the TEII _srf or ybgC been graphically describes how the time of cloning.

Cloning details:
Construction of plasmid pUC19act This basis is the plasmid pUCadc_ctfA / B_thlA with SEQ ID NO: 14, which is a vector of clostridial genes for acetoacetate-decarboxylase (adc), CoA-transferase (ctfA / B) and thiolase (thlA) It was cloned in pUC18. The gene was cloned in the opposite direction relative to the lacZ-gene. The gene can therefore be transcribed under the control of the lac-promoter and the adc_ctfA / B_thlA fragment (3311Bp) was again cut with the restriction endonucleases SalI and EcoRI and ligated in the vector pUC19 which had been cut with the same enzymes ( Reply drying kit according to manufacturer's instructions; Fermentas GmbH, St. Leon-Rot). For every 10 μl of ligation reaction, 100 μl of CaCl ₂ -competent E. ColiXL1-B-cells were used for transformation. The resulting clone was subsequently grown in LB-ampicillin-liquid medium and the plasmid-DNA was isolated. The isolated plasmid was examined by restriction analysis (SalI / EcoRI). The plasmid having SEQ ID NO: 12 was marked with pUC19atc and was subsequently used for transformation in various E. coli strains, which were subsequently tested for the formation of acetone. In this case, the construct containing the clostridial gene forming acetone was used as a comparison with the rest of the construct.

Construction of plasmid pUC18att:
This basis is the plasmid pUCadc_ctfA / B_thlA with SEQ ID NO: 14, which clones the clostridial gene for acetoacetate-decarboxylase (adc), CoA-transferase (ctfA / B) and thiolase (thlA) in the vector pUC18 It has become.

Thioesterase II- gene TEII _srf, was amplified by primers TeIIpUCBamfw (CAA TTG GGA TCC GAT AAC AAT TTC ACA CAG) and TeIIpUCAccrev (GAG ATC TGG TAC CCG GTT AAA TGA TCG GA) plasmid PTEIIsrf PCR of using. In this case, the primer mediates the incorporation of a BamHI-cleavage site at the 5′-end of the amplified fragment or an Acc65I-cleavage site at the 3′-end. Synergy-polymerase (GeneCraft GmbH, Ludinghausen) was used according to the manufacturer's instructions under the following conditions:

A fragment of the expected size of about 800 Bp was obtained. This was first subcloned into pJET-vector (Fermentas, St. Leon-Rot) according to the manufacturer's instructions (pJet_thIIpUC). Subsequently, ThII from this plasmid _srf - fragment again cut with the restriction end wetting over ase BamHI and Acc65I the (776Bp), and gel-eluted (Gel Extraction Kit, Peqlab Biotechnologie GmbH, won in accordance with the manufacturer's instructions). The vector pUCadc_ctfA / B_thlA was similarly treated with restriction enzymes BamHI and Acc65I to extract a ctfA / B-fragment (1331Bp). The reaction appeared in the agarose gel and the band was gel eluted with the remaining vector (4633Bp) (gel extraction kit, peqlab Biotechnologie GmbH, acquired according to manufacturer's instructions). Subsequently, ligation of the vector and the fragment was carried out using a rapid ligation kit (Fermentas GmbH, St. Leon-Rot) according to the manufacturer's instructions. For every 10 μl of ligation reaction, 100 μl of CaCl ₂ -competent E. ColiXL1-B-cells were used for transformation. The resulting clone was subsequently grown in LB-ampicillin-liquid medium and the plasmid-DNA was isolated. The isolated plasmid was examined by restriction analysis (BamHI / Acc65I). The resulting plasmid pUC18att_sub was further used for thIA-fragment and thiolase-promoter cloning.

The thiolase gene thlA including the thiolase-promoter was amplified by PCR of genomic DNA derived from C. acetobutylicum. Primers used for PCR, PthlApUCSalfw (CAT GAT TTG TCG ACG GTT AGC ATA TG) and PthlApUCBamrev (CAG AGT TAT TTT TAA GGA TCC TTT CTA GC) are used at the 5'-end of the amplified fragment or BamHI Mediates the incorporation of a cleavage site at the 3′-end. Taq-master mix (Qiagen, Hilden) was used according to the manufacturer's instructions with the addition of 2.5 mM MgSO ₄ under the following conditions:

A fragment of the expected size of about 1400 Bp was obtained. This was first subcloned into pJET-vector (Fermentas, St. Leon-Rot) according to the manufacturer's instructions (pJet_pthlApUC). Subsequently, thlA from this plasmid _srf - fragment again cut with the restriction end wetting over ase SalI and BamHI (1397Bp), and gel-eluted (Gel Extraction Kit, Peqlab Biotechnologie GmbH, won in accordance with the manufacturer's instructions). A thiolase-fragment (no promoter, 1217Bp) was excised from the plasmid pUC18att_sub constructed in the front part with SalI and BamHI, the remaining vector (4192Bp) was gel-eluted, and the thiolase fragment (1397Bp) containing the promoter was ligated ( According to the instructions of the manufacturer of the rapid ligation kit; Fermentas GmbH, St. Leon-Rot). For every 10 μl of ligation reaction, 100 μl of CaCl ₂ -competent E. ColiXL1-B-cells were used for transformation. The resulting clone was subsequently grown in LB-ampicillin-liquid medium and the plasmid-DNA was isolated. Isolated plasmids were checked by restriction analysis (SalI / BamHI) and sequenced (Agowa GmbH, Berlin). This plasmid was marked with pUC18att (SEQ ID NO: 10) and used in various E. coli strains for further transformation, which was later tested for the formation of acetone.

Construction of plasmid pUC19att This basis is plasmid pUC19act, which is a clone of the clostridial gene for acetoacetate-decarboxylase (adc), CoA-transferase (ctfA / B) and thiolase (thlA) in vector pUC19 It is.

TEII _srf - fragments, restriction enzymes BamHI and Acc65I cut from pJet_teIIpUC in (776Bp), and gel-eluted (Gel Extraction Kit, Peqlab Biotechnologie GmbH, won in accordance with the manufacturer's instructions). The vector pUC19act was cleaved with the same enzyme, and the ctfA / B fragment was removed. After appearance in agarose, the remaining vector band (4633Bp) was gel eluted (gel extraction kit, peqlab Biotechnologie GmbH, obtained according to manufacturer's instructions). Subsequently, the vector fragment and the teIIpUC-fragment were ligated. For this, a quick ligation kit (New England Biolabs GmbH, Frankfurt am Main) was used according to the manufacturer's instructions. Transformation was performed as described above. In order to examine the resulting clones, they were subsequently grown in LB-liquid medium and the plasmid-DNA was isolated. The isolated plasmid was examined by restriction analysis (BamHI / Acc65I). The plasmid thus obtained was marked with pUC19att (SEQ ID NO: 9) and used in various E. coli strains for further transformation, which was later tested for the formation of acetone.

Construction of plasmid PUC19ayt For this purpose the plasmid pUC19att was formed.

Genomic DNA PCR using the ybgc gene of ybgcpUCBamfw (CTC TAG AAG GAT CCT GTT TAA CTT TAA G) and ybgcpUCAccrev (ATT GGG TAC CTC ATT GCA TAC TCC G) Amplified by In this case, the primer mediates the incorporation of a BamHI-cleavage site at the 5′-end of the amplified fragment or an Acc65I-cleavage site at the 3′-end. Taq-master mix (Qiagen, Hilden) was used according to the manufacturer's instructions under the following conditions:

  A fragment of the expected size of about 490 Bp was obtained. This was first subcloned in pJET-vector (Fermentas, St. Leon-Rot) according to the manufacturer's instructions (pJet_ybgcpUC). Subsequently, the ybgc-fragment was cut from this plasmid again with restriction endonucleases BamHI and Acc65I (465 Bp) and gel eluted (gel extraction kit, peqlab Biotechnologie GmbH, obtained according to manufacturer's instructions).

The vector pUC19att was treated in the same restriction enzymes BamHI and Acc65I, teII _srf - fragment (468Bp) was obtained. The reaction appeared in the agarose gel and the band was gel eluted with the remaining vector (4633 Bp) (gel extraction kit, peqlab Biotechnologie GmbH, acquired according to manufacturer's instructions). Subsequently, ligation of vectors and fragments was performed using a rapid ligation kit (New England Biolabs GmbH, Frankfurt am Main) according to the manufacturer's instructions. For every 10 μl of ligation reaction, 100 μl of CaCl ₂ -competent E. ColiXL1-B-cells were used for transformation. The resulting clone was subsequently grown in LB-ampicillin-liquid medium and the plasmid-DNA was isolated. The isolated plasmid was checked by restriction analysis (BamHI / Acc65I) and sequenced (Agowa GmbH, Berlin). The resulting plasmid was marked with pUC19ayt (SEQ ID NO: 11) and used with various E. coli strains for further transformation, which was later tested for the formation of acetone.

  A summary of the plasmid constructs is summarized in Table 1.

Table 1: Plasmid constructs Plasmid size (Bp) Insert promoter
_{pSKatt 5771 teII srf (adc, thlA} ) lac
_{pKSatt 5925 teII srf (adc, thlA} ) thl
_{pUC19att 5409 teII srf (adc, thlA} ) lac
_{pUC18att 5589 teII srf (adc, thlA} ) thl
pUC19ayt 5101 ybgC (adc, thlA) lac
pUC19act 5964 ctfA / B (adc, thlA) lac

Example 5: Acetone formation in E. Coli All plasmid variants obtained (see Table 1) were tested for acetone formation in E. Coli-clonal strain HB101. The analysis was carried out on a 5 ml basis in ampicillin-containing LB medium (100 μg / ml) after transplanting from the corresponding preculture and incubating at 37 ° C. and 200 rpm. The optical density (600 nm) is observed by optical analysis, and when the value is about 0.5 to 0.6, expression of the cloned gene can be confirmed by adding 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside). Induction and glucose (20 g / l) was added 3-4 hours later. No induction was performed with mutants that controlled the thiolase-promoter. This is because structural expression was performed.

  Probes were removed at specific time points over a period of about 48 hours and the concentrations of acetone and acetate were determined in the cell-free medium supernatant by gas chromatography. Optionally, glucose was further added in various amounts (within a range of 10-40 g / l) 4 hours after or simultaneously with the induction.

  In E. Coli HB101, significant acetone production was detected in all plasmid variants. FIG. 11 exemplarily shows a growth curve with the amount of acetone and the amount of acetate formed for each mutant. A summary of the results using the maximum acetone concentration is shown in Table 2. FIG. 11 shows the optical density (600 nm) and the acetone concentration and acetate concentration (mM) at the selected time point, respectively. The black arrow indicates the time when IPTG (1 mM) was added, and the green arrow indicates the time when glucose (20 g / l) was added.

Table 2: Summary of maximum acetone concentration of each plasmid variant in 5 ml medium Medium Plasmid insert promoter Maximum acetone concentration, strain, medium
_{pSKatt teII srf (adc, thlA)} lac 34mM (2.0 g / l, HB101, LB Amp)
_{pKSatt teII srf (adc, thlA)} thl 36 mM (2.1 g / l, HB101, LB _Amp )
_{pUC19att teII srf (adc, thlA)} lac 30 mM (1.7 g / l, HB101, LB _Amp )
_{pUC18att teII srf (adc, thlA)} thl 35 mM (2.0 g / l, HB101, LB _Amp )
_{pUC19ayt ybgC srf (adc, thlA)} lac 60 mM (3.5 g / l, HB101, LB _Amp )
_{pUC19act ctfA / B srf (adc,} thlA) lac 46 mM (2.7 g / l, HB101, LB _Amp )

Example 6: Comparison of the acetone pathway using the cloned ybgC-gene derived from Haemophilus influenzae and the acetone pathway derived from clostridial alone In subsequent experiments, 100 ml-medium (LB _Amp ) with mutant pUC19ayt and pUC19act as controls was also obtained. The results obtained were reproduced and examined (37 ° C., 200 rpm). The main medium was inoculated with 1 ml of the corresponding preculture and induced with 1 mM IPTG at an OD _{600 nm} of 0.5. Unlike previous tests, the addition of glucose (20 g / l) was repeated 18 hours after the first addition. In addition, in parallel with each time the probe was removed, the glucose content of the culture was measured so that the glucose consumption could be calculated. Glucose was measured by an optical-enzymatic test by Bergmeyer (1970). At that time, the reaction of glucose to 6-phospho-gluconate and NADPH was carried out in two steps with the addition of ATP and NADP ⁺ by enzyme hexokinase and glucose-6-phosphate-dehydrogenase (Bergmeyer, HU ( Editor-in-Chief: Methods of Enzymatic Analysis, 2nd edition, Verlag Chemie, Weinheim Deerfield Beach-Basel 1970). In this case, the amount of NADPH formed was proportional to the amount of glucose present and could be optically determined by the change in absorbance at a wavelength of 340 nm. This result is shown in FIGS. 12A and 12B. It can be seen that the glucose in culture A (E. Coli HB101 pUC19ayt) has already been completely consumed by the time of the second addition.

  However, there was no sign of further increase in the amount of acetone even when glucose was newly used, and it was not consumed. This is because the decrease could not be clearly confirmed. This stems from the fact that other factors have already limited growth by this point. This is because there is no further increase in optical density. A maximum of 67 mM acetone (3.9 g / l) was formed. Similarly, a culture B having a control plasmid pUC19act carried out under the same conditions was prepared. Although glucose was not completely consumed by the time of the second addition, the relationship between the administration of new glucose and the increase in the amount of acetone formed remained unaffected. The maximum amount of acetone was 20 mM (1.2 g / l). Therefore, the mutant with the cloned ybgC-gene derived from Haemophilus influenzae produced three times more acetone under this condition than the control culture with only the clostridial gene.

  FIG. 12 shows the optical density (600 nm), acetone concentration and acetate concentration (mM), and glucose content (g / l) at the selected time point. The black arrow means the time when IPTG (1 mM) was added, and the green arrow means the time when glucose (20 g / l) was added; top: E. Coli HB101 pUC19ayt, bottom: E. Coli HB101 pUC19act ( Control).

Claims (8)

Next method steps:
A: a step of enzymatically reacting acetyl-CoA to acetoacetyl-CoA, wherein the reaction has ketothiolase activity and has an amino acid sequence that is at least 90% identical to amino acid SEQ ID NO: 16 A step catalyzed by
B: A step in which acetoacetyl-CoA is enzymatically reacted to acetoacetate and CoA, the reaction comprising acetoacetate-CoA-hydrolase, acyl-CoA-thioesterase, acyl-CoA-synthase or acyl A step catalyzed by CoA-thiokinase,
C: acetoacetate decarboxylated comprising the steps of acetone and CO _2, catalyzed by a polypeptide having an amino acid sequence identical having acetoacetate decarboxylase activity, and an amino acid sequence numbers 18 to at least 90% Process
A process for preparing acetone starting from acetyl-Coenzyme A having:
In method step B, coenzyme A is not diverted to the receptor molecule,
The enzymatic reaction in method step B is catalyzed by a polypeptide having acetoacetate-CoA-hydrolase activity and having an amino acid sequence that is at least 90% identical to amino acid SEQ ID NO: 5 A process for producing acetone.   The process according to claim 1, wherein the reaction in process step C takes place spontaneously.   The method according to claim 1 or 2, which is carried out by a microorganism. Microorganisms are at least:
a nucleic acid encoding a protein of at least one polypeptide that catalyzes the reaction in method step A), and b has acetoacetate-CoA-hydrolase activity and is at least 90% identical to amino acid SEQ ID NO: 5 A nucleic acid encoding a protein of a polypeptide having an amino acid sequence, and c. A nucleic acid encoding a protein of at least one polypeptide that catalyzes the reaction in method step C), wherein the nucleic acids a, b and c are in a microorganism. 4. The method of claim 3, wherein the method is used to ensure expression of a polypeptide in Nucleic acid a is:
aa DNA sequence set forth in SEQ ID NO: 17 or a complementary strand thereof,
bb a DNA sequence that hybridizes under stringent conditions to the DNA sequence described in aa ;
Pressurized et chosen method of claim 4. Nucleic acid c is:
dd DNA sequence set forth in SEQ ID NO: 19 or its complementary strand,
ee a DNA sequence that hybridizes under stringent conditions to the DNA sequence described in dd ;
Pressurized et chosen method of claim 4 or 5. Nucleic acid b is:
mm DNA sequence set forth in SEQ ID NO: 6 or its complementary strand,
nn a DNA sequence that hybridizes under stringent conditions to the DNA sequence described in mm ;
Pressurized et selected, method according to any one of claims 4 to 6. Nucleic acids a, b and c are on one nucleotide chain and this is:
Plasmid pSKatt having the nucleic acid sequence according to SEQ ID NO: 7,
Plasmid pKSatt having the nucleic acid sequence according to SEQ ID NO: 8,
Plasmid pUC19att having the nucleic acid sequence according to SEQ ID NO: 9,
Plasmid pUC18att having the nucleic acid sequence according to SEQ ID NO: 10,
Plasmid pUC19ayt having the nucleic acid sequence according to SEQ ID NO: 11
It is selected from the group comprising A method according to any one of claims 4 to 7.

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